Use of fructose-1,6-diphosphate for treating myocardial infarction

ABSTRACT

The invention relates to methods of treatment of patients with heat disorders using fructose-1,6-diphosphate.

This is a continuation of co-pending application Ser. No. 07/177,617,filed on Apr. 5, 1988, now abandoned, which is a continuation ofapplication Ser. No. 06/784,381, filed Oct. 4, 1985, now U.S. Pat. No.4,757,052, which is a continuation of application Ser. No. 06/414,551,filed Sept. 3, 1982, now U.S. Pat. No. 4,546,095, which is acontinuation of application Ser. No. 06/170,614, filed July 21, 1980,and now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the methods of treatment of patientswith heart disorders and more particularly to the method of usingFructose-1,6-Diphospate in treatment of the above mentioned diseases,and also as a protective agent against unforeseen catastrophichypotension or hypoxia during operative procedures, and as apreservative agent for transplantation organs.

2. General Background and Prior Art

In medicine and physiology it is well-known that a continuous supply ofenergy is necessary for the function and maintenance of a living stateby cells. The degree of intracellular energy is measured by the ratio ofhigh energy phosphate compounds to those of less energy potential (i.e.,adenoisine triphosphate to adenosie diphosphate and adenosinemonophosphate). The biochemical pathways which produce high energyphosphate compounds have been well established in the scientificliterature as a chain of reactions that result in the breakdown of themajor substrates, glucose or other sugars to pyruvic and lactic acidsand is a process of carbohydrate metabolism. Although one stage ofglycolysis requires oxidation by dehydrogenation, this may beaccomplished without oxygen, so the process as a whole may be anaerobic.The pyruvic acid formed by glycolysis is then oxidized to carbon dioxideand water. This oxidation is the source of most of the utilizable energy(ATP) derived from carbohydrate metabolism. Glycolysis also yields someenergy in the form of ATP which can be utilized for muscle contractionand other functions. This is particularly important during suddenstrenuous exercise, when energy must be made available in excess of thatwhich can be provided by oxidation processes.

The glycolytic process taking place in animal tissues involves thesequence of intermediates: ##STR1## fructose -1,6 - diP→glyceraldehyde -3-P+dihydroxyacetone-P [→glyceraldehyde - 3-P]→3 phosphoglyceric acid →2 phosphoglyceric acid→phosphoenolpyruvic acid→pyruvic acid→lactic acid

Fructose - 1,6 - diP is cleared by the enzyme aldolase between the thirdand fourth carbon atoms to form two triose phosphate molecules,glyceraldehyde - 3-P and dihydroxyacetone phosphate. ##STR2##

This reaction is reversible. Glyceraldehyde - 3-P and dihydroxyacetone -P are freely interconvertible through the action of triose-P-isomerase.

The next step in the main stream of glycolysis consists of the combinedphosphorylation and oxidation of glyceraldehyde - 3-P to 1,3diphosphoglyceric acid, which is catalyzed by the enzymeglyceraldehyhde - 3-P dehydrogenase: Glyceraldehyde -3-P+Pi+DPN+⃡1,3Diphosphoglyceric acid+DPN . N+H⁺

The conversion of glyceraldehyde - 3-P to 1.3 diphosphoglyceric acidproceeds anaerobically through oxidation by DPN⁺. In this process DPN⁺is converted to DPN.sup.. H, and the reaction would soon cease without amechanism to reoxide DPN.sup.. H. In this process DPN⁺ is converted toDPN.sup.. H, and the reaction would soon cease without a mechanism toreoxide DPN.sup.. H to DPN⁺, since the amount of coenzyme present isvery small. Anaerobically DPN.sup.. H is oxidized to DPN⁺ by pyruvicacid, with the formation of lactic acid: ##STR3##

Substances other than pyruvic acid may serve also to oxidize DPN.sup.. Hto DPN⁺. Among these are dihydroxyacetone-P, which is reduced to2-glycerophosphate, and oxaloacetic acid, which is reduced to malicacid. Reduction by these substances is of importance in starting theglycolytic process before sufficient pyruvic acid has been formed tofunction in the regeneration of DPN⁺. No ATP is formed in the oxidationof DPN.sup.. H by pyruvic acid.

When the supply of oxygen to the tissues and the oxidative mechanismsare adequate, the DPN⁺ H is oxidized to DPN⁺ through the mitochondrialelectron transport chain: DPN.sup.. H - FP - Cytochromes - O₂ →DPN³⁰ +H₂O+3ATP

Consequently, lactic acid accumulates in tissues only when oxidation byO₂ cannot keep up with glycolytic reactions and pyruvic acid is reducedto lactic acid.

In this stage of glycolysis we have the first generation of utilizableenergy as ATP. 1 molecule of ATP per triose phosphate mol when DPN.sup..H is oxidized anaerobically (by pyruvate), and 4 mols per triosephosphate mol when DPN.sup.. H is oxidized by O₂.

The next stage of glycolysis consists in the conversion of3-phosphoglyceric acid to 2-phosphoglyceric acid by the enzymephosphoglyceromutase, which requires catalytic amounts of 2,3 -diphosphoglyceric acid.

Glycolysis proceeds by the conversion of 2-phosphoglyceric acid tophosphoenolgyruvic acid through action of the enzyme enolase. Thisreaction involves dehydration of 2-phosphoglyceric acid and is freelyreversible. The loss of water converts the low-energy phosphate group of2-phosphoglyceric acid to the high energy phosphate group ofphosphopyruvic acid. Because of the high energy phosphate group presentin phosphopyruvic acid, it reacts with ADP to form ATP and pyruvic acidto complete glycolysis properly. The reaction is catalyzed by the enzymeATP-phosphopyruvic transphosphorylase or pyruvic kinase, which requiresMg⁺⁺ and K⁺ for activation. This reaction accounts for the formation of2 mols of ATP per mol of sugar glycolyzed.

The reactions of glycolysis in animal tissues lead to the end productspyruvic and lactic acids. Pyruvic acid is oxidized and converted toacetyl CoA by an oxidative 2-ketodecarboxylase enzyme. Oxidation of theDPN.sup.. H in the electron transport chain yields 3ATP per mol. Theacetyl CoA formed from pyruvic acid is oxidized in the citric acid cycleto CO₂ to H₂ O with the formation of 12ATP per mol. So the aerobicpathway for the breakdown of sugars and fatty acids is dependent upon aready supply of oxygen, which is provided to the body tissues throughthe bloodstream and is bound weakly to hemoglobin. Thus, interruption ofeither the pumping action of the heart occlusion of one of the arteriesor failure to oxygenate the blood being circulated by the lungs willresult in either a regional or generalized unavailability of oxygen.

If oxygen is not supplied to living tissue for any of the abovementioned reasons, aerobic or oxygen dependent metabolism ceases. Thisleads to an attempt to compensate the failure in oxygen supply by anincrease in the rate of anaerobic metabolism.

It was already noted that the anaerobic metabolic pathway orcarbohydrates involves glucose which then becomes phosphorated (hassix-carbon sugar). These molecules than break down to trioses(three-carbon sugars) and enter the aerobic pathway as the pyruvatemolecule. In tissues that have limited blood supply or, for some otherreason fail to be given an adequate amount of oxygen, the aerobicpathway must provide all of the energy necessary for cellular function.However, during any form of oxygen deprivation, whether it is from heartattack or from blood loss, leading to hypoperfusion as in an injury, thepathways of metabolism are refractory to the further entrance of theglucose into the cell, and the critical breakdown point in the metabolicpathway is at the phospho-fructo-kinase enzyme step. This means thatunless something different is done, the individual deteriorates to thepoint where his tissues are unable, because of the injury, to regainfunction which may lead to a fatal outcome.

Investigations have been made on the effect of sugars on the recovery ofheart functions. For example, Dr. Pasi Kettunen of Finland described hisexperiments on the "Comparison of the Effect of Glucose and Fructose onthe Recovery of the Heart Preparation" (Scand, j. clin. Lab. Invest. 37,705-708, 1977). Potassium citrate solution was used for heart arrest,and heart function was recovered by infusion of Locke's solution, plusglucose, fructose or sucrose. During recovery period, the amplitude andfrequency of heart beats, the lactic acid in the drained perfusionsolution, pH and potassium concentration were measured. The use ofglucose, fructose or sucrose made no significant difference to any ofthese parameters. Next, the metabolism of glucose and fructose in theheart was investigated and on a metabolic basis the use of glucose forresuscitation would seem to be more appropriate than fructose.

Dr. L. H. Opie and P. Owen in their investigation of glycolysis in acuteexperimental myocardial infarction found out that glycolysis duringanaerobic circumstances may be accelerated by all the factors thatstimulate phosphofructokinase activity. They indicated that there is anoverwhelming change that may be expected to inhibit phosphofructokinaseactivity, namely the intracellular accumulation of hydrogen ions. Thisphenomena was confirmed by Ui in 1960 and Kubler and Piieckermann in1970. Dr Opie then wrote that some index of phosphofructokinase activitycan be obtained by measurements of tissue contents ofglucose-6-diphosphate. Glycolytic flow may be compared to a regularstream, phosphofructokinase acts as a am-wall, inhibiting the flow ofglycolysis. Thus it is evident that these investigations, being valuableby themselves could not provide a sufficient method and an agent whichcan modify energy requirements intracellularly in the fact of low oxygenlevels, poor blood circulation of poor distribution of circulation.

3 GENERAL DISCUSSION OF THE PRESENT INVENTION

As it has been mentioned above, the biological attempt to compensate forinsufficiency of aerobic metabolism leads to a temporary increase in therate of anaerobic metabolism, but this compensatory mechanism is limitedby acidosis of the involved tissues. Once the inevitable acidosisoccurs, this pathway is inactivated also. The premature shutdown of theanaerobic pathway is not an irreversible phenomena, rather the effect ofacidosis from lack of oxygen causes interruption at several criticalsteps. First, glucose cannot gain access to the cellular interior. Next,phosphofructokinase, the enzyme which catalyzes the conversion offructose, 6, monophosphate to fructose 1,6, diphosphate is renderedinactive.

To bypass these metabolic bottlenecks, fructose 1,6 diphosphate can beinjected in amounts exceeding substantially that which could beavailable in the natural state. This sugar, if metabolized to lacticacid, will produce 4 molecules of ATP without the requirement of oxygen.Besides, the fructose molecule unlike glucose does not require energy tocross the cell membrane and is not dependent upon the action of insulinand it enters above the PFK enzyme level which has been damaged duringthe ischemic process. Therefore, the addition of fructose 1,6diphosphate appears to be a significant step in by-passing this andobtaining temporary energy to sustain ischemic tissues, perhaps even thewhole organism over a limited time while it is being infused. It mayproduce from 20 to 80 percent of the energy required for a given tissue.In a number of experiments which has been carried out by Dr. AngelMarkov, in the Department of Medicine at the University of Mississippi,it has been shown repeatedly that the material is useful when given inhemorrhagic shock, experimental myocardial infarction, and otherconditions where tissue is without oxygen. In Dr. James W. Jones'experiments, it has appeared that this material is very useful inmetabolism of the heart. The research gives an evidence that thematerial could be useful in treating patients with the followingdisorders;

1. Hemorrhagic shock;

2. Cardiogenic shock;

3. Cyanide poisoning and any poisoning of oxidative metabolism;

4. Myocardial preservation;

5. Therapeutic agent after myocardial infarction;

6. During respiratory failure with low blood oxygen levels;

7. During operative procedures as a protective agent against unforeseencatastrophic hypotension or hypoxia;

8. As a preservative agent for transplantation organs such as a kidney,liver, heart, etc.;

9. As an agent to enhance drugs it would be given as chemotherapeuticagents to destroy a tumor cell;

10. Sickle-Cell anemia;

11. Reversal barbituate overdose;

12. Blood preservation;

13. As an agent for treating disorders in white blood cells'phargocytosis; and

14. Endotoxin shock.

In the experiments regarding an irreversible hemorrhagic shock, resultswere obtained in the dog model using Wigger's modified technique. Wecompared a group of animals receiving IV administration offructose-1,6-diphosphate (FDP) to a group receiving equimolar glucose.

The metabolic changes resulting from generalized tissue hypoxia revealedalteration in the acid base balance and increased lactic acidconcentration in blood reflecting the relatively anaerobic character ofthe metabolism. The rater of anaerobic glycolysis for different tissueappears to be a direct function of the severity of hypotension and thesevere hypotension could accelerate glycolysis in the heart and otherorgans by 100% when compared to control. Although there is an initialincrease of anaerobic glycolysis after a certain period, its rate beginsto decline due to progressively increasing acidosis secondary toincreased lactemia.

The phosphorylation of fructose-6-phosphate is an important controlpoint in the Embden-Meyerhof pathway. Phosphofructokinase (PFK) is amultivalent enzyme which catalyzes this rate limiting reaction. PFK isstimulated by ADP, AMP and FDP and inhibited by ATP, citrate andacidosis. It is already known that the ischemic inhibition of glycolysisis due to inactivation of PFK by progressively increasing intracellularacidosis. As in shock severe metabolic acidosis takes place, it isreasonable to assume that PFK is inactivated. This is substantiated bythe observation that in hemorrhagic shock plasma lactate initiallyincreases very rapidly and then lactate production appears to decline.In our experiments the plasma lactate measured at 2 hrs 45 min after theonset of hypotension in the controls was found to be 76±14 mg %, whilein dogs treated with FDP that concentration was in the order of 124±16mg %.

Out experiments also gave evidence that serious cardiac failure(secondary to impaired energy production occurs approximately at thetime when hemorrhagic shock becomes irreversible and it is probablyresponsible for the pathogenesis of hemorrhagic shock is a complexphenomenon in which every structural unit in the entire organism isaffected (to different degrees). Yet, the circulatory weak spotcontributing to the irreversibility of the hemorrhagic syndrome appearsto be the heart itself. Forty years ago Wiggers realized that in thecourse of hemorrhagic shock, a deficit in coronary blood flow thatarises could be implicated as a major cause for the irreversibility ofthe condition.

In control animals during the late course of the hemorrhagic shockchanges were observed in metabolism in the endocardium taking place tobe similar to those observed in acute myocardial ischemia. These aremanifested by ST segment elevation which occurred in all non-treatedanimals and depletion of ATP and CP in the endocardium to the samedegree as found in acute myocardial infarction.

To allow for a broader and more complete interpretation of the results,a small paragraph will be devoted to the ability of FDP to cross thecellular membranes. Although the actual mechanism of trans-membranouspassage of FDP is not known, there is direct and indirect evidence whichsubstantiate that it crosses through cellular membranes. These data areconsistent with the modern concept of the membrane which is capable oftransferring high energy substrate through a series of coupledreactions. From experimental data, indirect evidence substantiates theassumption that FDP crosses the cellular membrane in different tissuesin animals and man. In isolated organs, for example, when FDP is addedperfusate, rabbit ileum increases the force of contraction, whileglucose-6-phosphate, fructose-6-phosphate or fructose and inorganicphosphate fail to produce the same effect. This effect of FDP on rabbitileum can be correlated with the increased availability of ATP and theregulatory effect of FDP on PFK, on pyruvate kinase and in inhibition of6-phosphogluconate dehydrogenase. As all of these enzymes are in thecytoplasm, FDP must cross the cellular membrane in order for such aneffect to be observed. Incubation of erythrocytes with 5% FDP causes alarge increase in their ATP and 2-3 DPG content. Equimolar glucose,glucose-6-phosphate, fructose-6-phosphate, fructose and inorganicphosphate failed to increase the ATP content in the erythrocytesincubated under the same conditions. Intracardiac administration of FDPin rat causes five times higher concentration of FDP in the lever thanin plasma 10 min after administration. Intravenous administration indogs of 5 g of FDP over 10 min causes the plasma lactic acid (measuredat one hour) to increase 21/2 times (from 6.13±1.7 to 16.45±2.16 mg %).In other experiments we have found that IV administration of FDP causesan increase of ATP, lactate, and FDP in all tissues that we havestudied, as well as increases in the trioses in the Embden-Meyerhofpathway.

We attempted to remove the inhibition of PFD, hence glycolysis, in theshock model described by intravenous administration of FDP, thusincreasing energy production, preventing cardiac damage and improvingsurvival. Fructose diphosphate in the described shock model reducedmortality to zero, prevented electrocardiographic ischemic changes andincreased significantly the ATP and CP in the endocardium. These changeswere associated with accelerated glycolysis seen by increased plasmalactate and myocardial tissue lactate.

From an energetic point of view the advantage of using FDP as theinitial substrate is that the net yield of the anaerobic metabolism ofone mole of glucose is 2 ATP, while if one mole of FDP is metabolized inthe same conditions it will produce 4 ATP because there is nophosphorylation of glucose and fructose-6-phosphate, reactions whichrequire utilization of ATP. On the other hand, while doubling thequantity of ATP produced, lactate production remains the same as thoughone mole of glucose had been metabolized. It is obvious that in order toobtain results in the direction of making usuable energy from FDP, it isnecessary to administer a large quantity. (The LD₅₀ of FDP whenadministered 500 mg/min in dogs is approximately 5.8-6 g/kg.)

Another important metabolic effect of FDP is that it causes asubstantial increase of ATP and 2-3 DPG in the erythrocytes. Thisincrease of 2-3 DPG is of importance for the oxygen exchange betweenhemoglobin and tissue. It may also contribute in part to energyproduction derived from the oxidative metabolism by increasing theoxygen availability to tissues in such a low flow state.

The significance of the present invention is not only limited to thesuccessful treatment of experimental irreversible hemorrhagic shock byincreasing metabolic activity of glycolysis, but as well it proposes aunifying concept for understanding the pathophysiological mechanism ofshock. There is no doubt that the nature of the initial insult isirrelevant to the genesis of shock, but what is important is the factthat the organism as a whole integral unit shares with its constituents(organs and cells) a common energy deficit. In the early stages of anyetiologic type of shock there is inadequate oxygen transport and supplyto tissue. Hence, there is an energy deficit. Every system, organ andindividual cell responds to this decrease in energy supply according totheir entropic state. As the biological system is an open system, anydeficit in free energy would be manifested by increasing the disorder ofthe system and as in shock the vicious cycle is initiated which leadsthe system to higher and higher degrees of disorder. The final stage ofthis intracellular disorder is the achievement of the same entropicstage between "milieu exterieur" and "milieu interieur" --defined inbiological terms as death.

It is thus an object of the present invention to provide a method oftreating patients during a hemorrhagic shock by fructose-1,6-diphosphate(FDP).

It is another object of the present invention to provide a method oftreating patients in cardiogenic shock with FDP.

It is a further object of the present invention to provide a method oftreating patients in case of cyanide poisoning by FDP.

It is another object of the present invention to provide a method ofmyocardial preservation by treating a patient with FDP.

It is a further object of the present invention to provide a method oftreating patients after myocardial infarction by using FDP as atherapeutic agent.

It is still another object of the present invention to provide a methodof treating patients with FDP during respiratory failure with low bloodoxygen levels.

It is a further object of the present invention to provide a method ofintroducing FDP during operative procedures so that it might be aprotective agent against unforeseen catastrophic hypotension or hypoxia.

It is still another object of the present invention to provide a methodof preservation of transplanted organs using FDP as a preservativeagent.

Still another object of the present invention is to provide a method oftreating cancer patients by using FDP as a possible means of causingdamage to the tumor cells and using it as a chemotherapeutic agent toenhance drugs in destroying tumor cells.

It is further an object of the present invention to provide a method oftreating patients with FDP during a sickle cell crisis.

Still another object of the present invention is to provide a method oftreating patients having a reversal barbituate overdose.

Another object of the present invention is to provide a method of usingFDP for blood preservation.

It is another object of the present invention to provide a method oftreating patients with disorders in white blood cells' phagocytosisusing FDP.

Finally, another object of the present invention is to provide a methodof treating patients with FDP during an endotoxin shock.

BRIEF DESCRIPTION OF THE DRAWINGS

Of further understanding of the nature and object of the presentinvention, reference should be had to the following detaileddescription, taken in conjunction with the accompanying drawings, inwhich like parts are given like reference numerals and, wherein:

FIG. 1 would illustrate mean arterial pressure responses of dogssubjected to hemorrhagic shock for three hours at 33 mm. Hg.;

FIG. 2(A-E) would illustrate the hemodynamic and electrocardiographiceffects of FDP when given to dogs with acute regional myocardialischemia at 45 minutes after the occlusion of a coronary artery;

FIG. 3 (A and B) would illustrate the adenosine triphosphate (ATP),creatine phosphate (CP) and tissue lactate content in the normallyperfused and ischemic cyocardium in both controls and FDP treated dogs;

FIG. 4 would illustrate the Adenyl nucleotide content in the myocardiumafter hypotension of 35 mm. Hg. for 3 hours;

FIG. 5 would illustrate the intestinal fluid loss and urine output ofdogs subjected to endotoxin shock;

FIG. 6-7 would illustrate the mean arterial pressure responses offifteen dogs that received LD₉₀ of endotoxin as a bolus IV injection andwere supported with fluid therapy at the same quantity as stated in FIG.1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The drug is a sugar diphospate compound that needs to be injectedintravenously in both a bolus form or a single dose or as multiplesingle doses or as a continuous infusion drip. The amounts to be givenshould not exceed 5 grams per kilogram body weight per hour in infusion.If the heart alone is having it infused then it would be per kilogramper tissue. This adjustment must be made because the drug hasdetrimental affects with a LD 50 or a lethal dose of 50% of the animalsgiven if this is exceeded. The dosages which have been useful are togive 50 ml per kilogram body weight in a single dose or to give 1.5 to 2ml per kilogram of body weight over each minute as in continuousinfusion.

We shall first discuss the method of treating of a hemorrhagic shock.The sequence of events following severe hemorrhage is believed to be:decreased venous return, decreased cardiac output, reduced arterialpressure, decreased blood flow to the organs with ensuing tissuehypoxemia. Hemorrhagic shock, therefore, represents acute circulatoryinsufficiency that leads to generalized tissue ischemia. Studiesdirected at the possible deleterious effects of the metabolicderangements or tissue products resulting from reduced flow in shockhave revealed alterations in the acid base balance, and chemicalcomposition of the blood reflecting the relatively anaerobic characterof metabolism in shock. The degree of metabolic acidosis, increasedplasma and tissue lactate in hemorrhagic shock are functions of theseverity of the hypotension. Although there is an increased rate ofanaerobic glycolysis and plasma glucose initially, after a certainperiod its rate begins to decline due to the progressively increasingacidosis caused by the increased lactate production. This progressiveinhibition of the Ebmden-Meyerhof process by acidosis appears to becaused by the inactivation of the pH acid) sensitive rate limitingenzyme of glycolysis, phosphofructokinase (PFK).

During the late phase of hemorrhagic shock, morphological and metabolicchanges that take place in the endocardium are similar to those observedin the course of acute myocardial ischemia. Cardiac failure may occur inhemorrhagic shock secondary to a deficit in coronary blood flow. Thehemorrhagic state becomes irreversible due to impairment of cardiacfunction. In view of this, the goal of the present invention wasconcerned with the evaluation of the therapeutic value offructose-1,6-diphosphate in preventing death and cardiac damage whenadministered during the oligemic phase of shock which has been shown tobe irreversible to the infusion of all shed blood. We regard this as themost valid technique for testing the efficacy of this agent.

The experiments were performed in b 25 pentobarbital anesthetized (30mg/kg IV) mongrel dogs of both sexes. After induction of anesthesia theanimals were incubated and ventilated with a Harvard respiration pumpwith the use of ambient air. Secured in the left lateral decubitusposition on the fluoroscopic table, a femoral artery was used tointroduce a large bore polyethylene catheter which was connected to a1.5 liter bottle elevated at 35 mm Hg above the atrial level. In theconnecting system an external calibrated electromagnetic flow probe(Statham) was incorporated in order to follow the direction and amountof blood flow. The pressure in this system was monitored through a sideopening with the aid of a Statham pressure gauge. The other femoralartery was percutaneously catheterized and a Judkins type pigtailcatheter was advanced to the aortic root. In the course of theexperiments this catheter was placed intermittently into the leftventricle when recordings were made. This set-up enable us to monitorboth aortic and left ventricular pressures via a single catheter. Thefemoral vein on the side where the incision was made was catheterized aswell as a superficial vein of the front limb transutaneously foradministration of the FDP, glucose, heparin and supplemental anesthetic.Parameters such as aortic, ventricular and femoral pressures, and EKGwere monitored on an Electronics for Medicine DR-8 recorder. Recordingof these parameters was made every 15 min. Heparin (6 mb/kg initiallyand thereafter 3 mg/kg/2 hrs) and antibiotics were given to all animalsthat were allowed to recover. A control period of one hour was allowedprior to bleeding the animal. During this period two arterial bloodsamples were drawn for pH, pO₂, pCO₂, pCO₂ combining power and lacticacid determinations. During the oliegemic phase four arterial sampleswere taken for determination of the same parameters described at 30 min,1 hr, 2 hrs, and 2 hrs 45 min.

The blood from the animals was withdrawn via the femoral artery (50-55ml/min) until the means arterial pressure dropped to 35 mm Hg and fromthen on the pressure was kept at this level for three hours. Theprotocol for evaluating the therapeutic effect of FDP was designed inthe following manner. The 11 control animals received an IV bolus of 500mg of glucose when the mean arterial pressure was stabilized at 35 mmHg, and thereafter, a constant infusion of 5% glucose was given at therate of 1.25 mg/kg/min. The treated dogs received FDP(fructose-1,6-diphosphate sodium salt, Sigma Chemical, Grade II) 500 mgas an initial bolus and constant infusion of a 5% solution at a rate of1.25 mg/kg/min. Both groups received an additional 500 mg IV bolus ofFDP or glucose every 30 min for the duration of the oligemic phase. Infive controls and six treated dogs the chest was opened at three hoursand with the aid of a specially designed cutting tool that had beencooled in liquid nitrogen, frozen transmural sections from the leftventricle were taken for ATP, creatine phosphate (CP), FDP and lacticacid determinations. The remaining 14 animals (6 controls and 8 treatedwith FDP) were retransfused at three hours and allowed to recover.

The epicardium and endocardium from the frozen tissue were separated(while still frozen) and homogenized in 6% perchloric acid. Thesupernatant was used to determine ATP, CP and lactate. The ATP andcreatine phosphate were assessed with the method described by Lamprecht(Lamprecht, W., and P. Stein. Methods of Enzymatic Analysis. Ed: H. V.Bergmeyer, New York, Academic Press, 1963, p. 610.) while lactate wasmeasured by the method of Marback (Marback, E. P., and M. H. Weil. Rapidenzymatic measurement of blood lactate and pyruvate. Clin. Chem. 13:314, 1967). Tissue content of FDP was assessed by the method of Bucherand Hohorst (Bucher, T., and H. J. Hohorst. Dihydroxyacetone phosphate,fructose 1-6 diphosphate and D-glyceraldehyde 3-phosphate. In. Methodsof Enzymatic Analysis. Ed: Bermeyer, New York, Academic Press, 1963,pgs. 246-252.)

In the initial phase of the hypovolemia there was continuous outflow ofblood into the reservoir. The level of blood was constantly monitored soit never exceeded 35 mm Hg above the atrial level. An overflow systemprevented the level of blood from rising too high. However, afterapproximately 11/2 hours the blood flow reversed itself spontaneously(from the bottle to the animal) in the animals of the control group. Inthe group treated with FDP, only two of them reversed their flow at 2hrs and 15 min and 2 hrs and 37 min. The amounts of blood uptake inthese animals were 24% and 17% respectively of the shed blood.

In both groups the heart rate increased. In the control group the heartrate exceeded 225 beats/min. while for the treated animals the heartrate returned faster towards control (after retransfusion) and did notreach 200 beats/min. In both groups a substantial metabolic acidosisdeveloped, though to a lesser degree in the control group. It was notexceptional in the group treated with FDP to find arterial pH of 6.8 andlower. The arterial plasma lactic acid concentration after the onset ofhemorrhage increased rapidly for both groups. However, after 2 hours inthe control group the lactic acids production began to level offcontrary to the group treated with FDP where the plasma lactatecontinued to rise. In the control group, the plasma lactateconcentration measured at 2 hours and 45 minutes was 76±14 mg %, whilein the dogs treated with FDP the plasma lactate at that time was in theorder of 124±16 mg %. The arterial pressure of the 14 animals that wereallowed to recover (6 controls and 8 treated with FDP) measured at 11/2hours after retransfusion demonstrated a striking difference.

All control animals demonstrated ischemic EKG changes on procordialleads (always between 55 min and 1 hours and 15 minutes after the onsetof shock). The ST elevation reached maximum at 2 hours (0.55±0.17 mV)and remained such until retransfusion when it began to decline. Incontrast, in the group that was treated with FDP, only minimal elevationwas observed in two animals. (N.B. In one of these dogs unintentionallyFDP was not administered until 30 minutes after the onset of thehypotension and in the other animal the infusion pump had stopped forapproximately 25 minutes.) In both animals after FDP was given theischemic changes disappeared. The endocardial ATP content in the groupthat received glucose fell by 50% while the creatine phosphate (CP) inthe epicardium and endocardium was 64% and 88% respectively less thannormal. The animals treated with FDP revealed normal ATP myocardialcontent and no transmural gradient; however, in the endocardium therewas 18% less CP than normal and no deficit in the epicardium. Theendocardial/epicardial tissue lactate ratio for the group that receivedglucose was 1.52±0.21, and for those treated with FDP, 2.26 ±0.26. All 6control animals that were retransfused died within 24 hours. All 8 dogstreated with FDP survived, had normal renal and bowel function and noneurological deficit was noted. All animals treated with FDP were keptfor six months after the experiments except for one which had to besacrificed at 11/2 months after the experiment because of a gangrenoushind leg on the side where the femoral artery was used for bleeding.

In FIG. 1, best shown are mean arterial pressure responses of dogssubjected to hemorrhagic shock for 3 hours at 35 mm Hg. The tests showedthat: (1) Irreversible experimental hemorrhagic shock of 3 hour durationat 35mm Hg AP could be successfully treated with FDP. (2) Intravenousadministration of FDP restores ATP and CP content in the myocardium,prevents the EKG ischemic changes and improves the myocardialcontractility. (3) FDP restores the depressed activity of glycolysis inthe endocardium induced by inactivation of PFK.

To evaluate the effect of FDP on global ischemia, 14 mongrel dogs wereplaced on cardiopulmonary bypass and their hearts were subjected tonormothermic arrest for one hour. Seven animals had 50 ml. of 5% glucosein 0.9% NaCl infused into the base of the aorta at 0 minutes, 20 minutesand 40 minutes following cross-clamping and, systemically, duringreperfusion. The remaining 7 animals were treated identically exceptthat 50 mg. of FDP was given instead of glucose and 2mg/kg/minute wasinfused systemically during reperfusion. Hemodynamic measurements weretaken before ischemia and at 15 and 30 minutes following reperfusionusing an isovolumetric technique.

    __________________________________________________________________________              Pre-Ischemia 15 minutes after                                                                            30 minutes after                                   Glucose                                                                              FDP   Glucose                                                                             FDP     Glucose                                                                             FDP                                __________________________________________________________________________    ML to LVEDP                                                                             51.4 ± 8.6                                                                        39.3 ± 3.2                                                                       23.3 ± 1.8                                                                       33.6 ± 3.7*                                                                        19.3 ± 1.3                                                                       36.1 ± 3.3**                    of 20 mmHg.                                                                   Peak      4894 ± 689                                                                        4710 ± 689                                                                       1126 ± 216                                                                       2242 ± 226**                                                                       1698 ± 350                                                                       3999 ± 404**                    dp/dt (mmHg/sec)                                                              Peak      144.6 ± 14.6                                                                      165 ± 12.9                                                                       36.9 ± 4.4                                                                       102.1 ± 11.5**                                                                     50.8 ± 6.3                                                                       125 ± 11.5**                    Effective pressure                                                            (mmHg)                                                                        Peak      66.4 ± 7.4                                                                        77.5 ± 4.6                                                                       17.8 ± 4.2                                                                       52.8 ± 5.4*                                                                        20.5 ± 2.9                                                                       61.4 ± 5.1**                    Mean pressure                                                                 (mmHg)                                                                        __________________________________________________________________________     *p 0.05                                                                       **p 0.01                                                                 

CONCLUSIONS: Intra-aortic and systemic administration of FDP has aprofound effect on myocardial performance following severe ischemia.Both contractility and compliance are vastly improved during therecovery period.

FIG. 2 summarizes the hemodynamic and electrocardiographic effects ofFDP when given to dogs with acute regional myocardial ischemia at 45minutes after the occlusion of a coronary artery. (The decimal pointsfor the left ventricular end-diastolic pressure are not visible;however, the scale is to 17.5 mm. Hg.) The FDP administration began at45 minutes after the onset of ischemia and continued throughout theexperiment as a constant infusion at the rate of 1.25mg./kg/min.

FIG. 3 shows the adenosine triphosphate (ATP), creatine phosphate (CP)and tissue lactate content in the normally perfused and ischemicmyocardium in both controls and FDP treated dogs. Note that FDP not onlycaused an increase of ATP and CP in ischemic myocardium, but also in thenormally perfused heart muscle. The same phenomenon was observed for thetissue lactic acid concentration.

FIG. 4 shows the Adenyl nucleotide content in the myocardium afterhypotension of 35 mm. Hg. for 3 hours. Note that in the epicardium inboth controls and those treated with FDP there is no significantdifference in ATP content while in the endocardium the ATP contentreached values compatible with acute myocardial ischemia. FDPadministration also prevents depletion of creatine phosphate (CP) inboth epicardium and endocardium.

To explain the mechanism of using FDP as an antidote for potassiumcyanide poisoning, we should note that potassium cyanide, or moreprecisely, the cyanide ion is a highly toxic substance which inhibitsspecifically the oxidation metabolism by inactivating the cytochrome a3in the electron transfer chain. IN such a circumstance the anaerobicglysolysis for a short period tries to compensate for the energy deficitby enhancing its activity. However, it can only partially meet theenergy demand of the organism. As it has already been specified, FDP isa high energy intermediate of the Emden-Meyerhoff pathway and when usedas an initial substrate in this metabolic pathway it will double itsefficiency. In dogs, 2 mg/kg IV bolus injection of potassium cyanide wasgiven and immediately they were treated with either FDP or Dextrose 5%.The dogs receiving 0.5 gm/kg over 30 minutes survived while the controlsreceiving the same amount of Dextrose died. Thus, it can be concludedthat FDP could be employed as an antidote for acute potassium cyanidepoisoning.

Referring now to the use of FDP as an anesthetic barbiturate it shouldbe specified that experimentally it has been demonstrated by Dr. SiesjoHarp, Jr. BK of the Netherlands that barbiturates when given inanesthetic concentrations cause a significant decline in carbohydrateutilization by the brain.

In our studies it was noted that the dogs receiving FDP (n=104) requiredtwice as much anesthetic to be maintained in a state of surgicalanesthesia as did the controls (n=103). Hence, the test was undertakento evaluate more objectively the analeptic effect of intra-arterialadministration of FDP on Surital (thiamylal)anesthesia in dogs.

In this paired test 10 dogs were anesthetized with Surital 35mg/kg IV.Within 10 minutes after induction of anesthesia, a #7 Sones catheter wasplaced in a common carotid artery via the femoral artery underfluoroscopic guidance. Injection of contrast media verified that theposition of the catheter did not obstruct the vessel, and portions ofthe brain were opacified transiently. Immediately following the contrastmedia injection the infusion of FDP or glucose was initiated (9-13 min.after induction of anesthesia). The FDP was prepared as a 5% solutionand infused at a constant rate of 1.91 ml/min. (97.5 mg/min.) into amain carotid artery. The controls received equal amounts of 5% dextrosevia the same route. The analeptic effect of FDP on signs, stages anddepth of anesthesia was evaluated by determining the time required forreappearance of eyelid reflex, opening of eyes, deglutiton, response topain, lifting of the head, sitting, and when the animal was able towalk. These data were compared with like date from the group thatreceived 5% dextrose.

The results showed that the time required for return of observableeyelid reflex after beginning the infusion into the carotid artery was3.70±0.67 min. in the FDP-treated dogs, while for the controls the timewas 70.00±25 min. (±SDM). The time required to regain waling ability wassignificantly shorter (76.6±16 min.) in the FDP treated dogs than in thedextrose controls (295.00±24.49 min.) (p<0.001). The table belowsummarizes the results.

    ______________________________________                                        Time in Minutes after Beginning of Infusion                                   Dog      Eyes     Response Lifts Able   Able                                  No.      Open     To Pain  Head  To Sit To Walk                               ______________________________________                                        FDP                                                                           1         7       17       28    48     60                                    2        14       19       33    47     70                                    3         5       10       14    35     50                                    4        12       18       19    52     95                                    5         4       15       20    40     82                                    GLUCOSE                                                                       1        80       120      170   300    310                                   2        75       130      135   225    300                                   3        105      160      190   310    330                                   4        54       125      165   215    280                                   5        40       100      120   310    265                                   ______________________________________                                    

Thus, it can be seen that intra-carotid administration of FDP to animalssubjected to thiamyulal anesthesia greatly reduces the time required forthe animal to regain consciousness and be able to walk. The sameanaleptic effect, but to a much lesser degree, is observed when FDP isadministered systemically. Since FDP has been shown to have profoundantishock activity in experimental animals and in man, that phenomenon -taken with the effects of FDP observed in this study - indicates thatFDP may have clinical potential in the treatment of barbiturateoverdose.

As the results of our tests showed, FDP can successfully be used forblood perservation.

Blood specimens taken from a number of dogs were mixed with FDP in thefollowing proportions: F1-25mg of FDP per 10ml of blood and F2-50mg ofFDP per 10ml of blood. The same proportion was prepared with glucose:G1-25mg of glucose per 10ml of blood and G2-50mg of glucose per 10ml ofblood. The blood specimens were refrigerated for 24 hours and then theblood was tested for ATP content after 24 hours 14 days. The table belowshows the results of the tests.

    ______________________________________                                        HOLE BLOOD                                                                    ______________________________________                                        Time/ATP F1              F2                                                   ______________________________________                                        24 hours 2.32 m mole/ml  3.34 m mole/ml                                       48 hours 2.34 m mole/ml  2.08 m mole/ml                                       7 days   Incubate 30' 37°                                                       0.70 m mole/ml  2.16 m mole/ml                                       9 days   0.82 m mole/ml  2.04 m mole/ml                                       ______________________________________                                        Time/ATP G1              G2                                                   ______________________________________                                        24 hours 0.22 m mole/ml  0.44 m mole/ml                                       48 hours 0.32 m mole/ml  0.34 m mole/ml                                       7 days   Incubate 30' 37°                                                       0.28 m mole/ml  0.18 m mole/ml                                       9 days   0.26 m mole/ml  0.40 m mole/ml                                       14 days  Incubate 40' 37°                                                       FDP 1.3 m mole/ml                                                                             Glucose 0.88 m mole/ml                                        No incubation                                                                 FDP 1.22 m mole/ml                                                                            Glucose 0.40 m mole/ml                               ______________________________________                                    

The tests were also conducted under the conditions when 25mg, 50mg, and100mg of FDP were mixed with 10ml of blood and 25mg, 50mg, and 100mg, ofglucose were mixed with 10ml of blood. After one night of refrigerationthe following results were shown.

    ______________________________________                                        Time/                                                                         ATP    FDP-25 mg    FDP-50 mg   FDP-100 mg                                    ______________________________________                                        24 hours                                                                             1.67 m mole/ml                                                                             2.2 m mole/ml                                                                             2.82 m mole/ml                                48 hours                                                                             1.92 m mole/ml                                                                             2.85 m mole/ml                                                                            3.40 m mole/ml                                ______________________________________                                        Time/                                                                         ATP    Glucose-25 mg                                                                              Glucose-50 mg                                                                             Glucose-100 mg                                ______________________________________                                        24 hours                                                                             0.475 m mole/ml                                                                            0.375 m mole/                                                                             0.250 m mole/ml                                                   ml                                                        48 hours                                                                             0.475 m mole/ml                                                                            0.450 m mole/                                                                             0.426 m mole/ml                                                   ml                                                        ______________________________________                                    

The tests were also conducted to investigate the effect of FDP on whiteblood cells (WBC) on metabolism in vitro. Well-known is the fact thatduring phagocytosis the energy expenditure of the white blood cellsincreases as they possess both metabolic pathways, i.e. oxydativemetabolism as well as anaerobic glycolysis. It was experimentallyassessed whether FDP can stimulate the carbohydrate metabolism of thesecells at rest. A similar phenomenon observed for normal humanerythrocytes, sickle cells and dog erythrocytes. From healthy humans anddogs, the white blood cells were separated and incubated at 37° Celsiusfor various periods of time with FDP, glucose and normal saline. At theend of incubation, ATP and intermediates of the glycolytic pathway wereassessed. The results indicated that the effect of FDP on carbohydratemetabolism for dog and human WBC is somewhat different. Nonetheless, theATP production in the WBC from human and canine blood when FDP is addedappears to be independent of the concentration (from 5 to 50 mg/ml), butrather a function of the incubation time. However, if AMP or ADP isadded to the samples, the increase of ATP becomes a function of the FDPconcentration. When human WBC are incubated with FDP the ATP content is2.47±0.25 m.mole/#WBC, while with the glucose incubated it was 1.28±0.21m.mole/#WBC. The pyruvic acid concentration in dog WBC incubated withFDP was 10 times greater than those incubated in glucose while those forthe human WBC, this difference was of much lesser degree (X2). Thedihydroxyacetone (DHA) in the human and canine WBC was 5 to 10 timesgreater in the FDP incubated than in the glucose ones.

In conclusion, FDP exerts profound stimulating effect on thecarbohydrate metabolism of human and canine WBC. This effect mightenhance the phagocytic activity of WBC as the latter do expend vastamounts of energy in that process. The eventual practical applicationwould be the use of FDP as a therapeutic agent in sepsis.

The study was also conducted to determine whether IV administration ofFDP to dogs subjected to lethal doses of endotoxin will prevent damageto the intestinal mucosa, and death. The rationale was that endotoxinper se and its deleterious effect on hemolate phase acidosis inhibitsglycolysis by inactivation of the rate limiting enzymephosphofructokinase.

Endotoxin shock was produced in digs by IV injection of E. coliendotoxin. Survival of control animals which received glucose and volumereplacement was 18%d while for those treated with FDP it was 90%.

FIG. 5 is a diagram showing the intestinal fluid loss and urine outputof dogs subjected to endotoxin shock. Seven dogs were treated withequimolar FDP and eight received glucose. All animals received fluidsupportive therapy (150 ml. of Rheomacrodex and 300 cc. of LactateRinger). In all controls there was blood in the fluid lost from theintestine while in the dogs treated with FDP no blood was observed.

Mean arterial pressure of the controls surviving 6 hours of observationwas 35%±7.21% below control, while in the FDP treated group it returnedto control values between 4 and 6 hours.

FIGS. 6∫7 show the mean arterial pressure responses of 15 dogs thatreceived LD₉₀ of endotoxin as a bolus IV injection and were supportedwith fluid therapy at the same quantity as stated in FIG. 1. Seven weretreated with FDP and the controls (8) received equimolar glucose.

It should be noted that FIG. 7 shows mean arterial pressure responses influid deprived dogs after IV injection of 1 mg/kg of endotoxin. In threepairs no fluid was given except for the FDP and glucose. The animalsthat received glucose died within 21/2 hours while the ones treated withFDP survived and are still living (five months after the experiment).

All controls had bloody intestinal fluid loss of 830±67 ml/6 hours,while in the treated group no blood was noted and fluid loss amounted to285±36 ml/6 hours. Urinary output of treated dogs was 276±98 ml/6 hoursand that of control group was 46 ±25 ml/6 hours. In the stomach andintestine of the control group hemorrhagic necrosis of the mucosa wasobserved while in the group treated with FDP only some vascularcongestion was noted. In three pairs no volume replacement was given.The three controls dies within 21/2 hours while the animals treated withFDP survived.

Administration of FDP to dogs subjected to lethal doses of endotoxinprevents death, intestinal hemorrhage and fluid loss, conserves normalrenal function and protects the mucosa of the stomach and intestine fromhemorrhagic necrosis.

The attenuation of digitalis toxicity can also be obtained by theadministration of FDP.

Dogs subjected to toxic doses of Digoxin were treated with FDP andglucose. FDP administration suppresses significantly the digitalisinduced dysarhythmia. Also the dogs treated with FDP survived, while thecontrols treated with Dextrose 5% died within 3 hours afteradministration of Digoxin. In this paired study 10 anesthesized dogsreceived 3 mg. IV Digoxin as bolus injection. Five of the digs received20 minutes after the digoxin injection 1 gm. of FDP and then on 2 mg/kgas a constant infusion for the next 3 hours. The five controls receivedin the same manner Dextrose 5%. The occurrence of dysarhythmia wassignificantly less in the group treated with FDP when compared to thecontrols. (Pi<0.001.). Moreover, no alternation in hemodynamicparameters such as cardiac output, arterial pressure were noted in theFDP treated group. In the control group prior to the death of theanimals there was decline in arterial pressure, vomiting, and perfuseddiarrhea.

Although the exact mechanism by which FDP attenutates the toxic effectof Digoxin cannot be explained at present, this observation might haveclinical application in the treatment of digitalis intoxication in man.

This invention provides a method of treatment of practically anyone whois suffering from an injury that had resulted in a loss of bloodsufficient to cause their body systems to have a low blood pressure.

Because many varying and different embodiments may be made within thescope of the inventive concept herein taught, and because manymodifications may be made in the embodiments herein detailed inaccordance with the descriptive requirement of the law, it is to beunderstood that the details herein are to be interpreted as illustrativeand not in a limiting sense.

What is claimed as invention is:
 1. A method of treating patients duringa hemorrhagic shock by introducing fructose-1,6-diphosphate in theamount of 50mg per kilogram of body weight in a single dose.
 2. Themethod of claim 1, wherein fructose-1,6-diphosphate is introduced in theamount of 1.,5 to 2mg per kilogram of body weight over each minute as incontinuous infusion.
 3. A method of treating patients suffering from acardiogenic shock by introducing fructose-1,6-diphosphate in the amountof 50 mg per kilogram of body weight in a single dose.
 4. The method ofclaim 3, wherein fructose-1,6diphosphate is introduced in the amount of1.5 to 2mg per kilogram of body weight over each minute as in continuousinfusion.
 5. A method of treating patients having cyanide poisoning byintroducing fructose-1,6-diphosphate in the dose of 100-250mg perkilogram of body weight in a single dose.
 6. The method of claim 5,wherein an hour later fructose-1,6-diphosphate is introduced in the doseof 2-3mg per kilogram of body weight over each minute as in continuousinfusion.
 7. A method of treating patients during respiratory failureand low blood oxygen levels by introducing fructose-1,6-diphosphate inthe amount of 50mg per kilogram of body weight in a single dose.
 8. Themethod according to claim 7, wherein fructose-1,6-diphosphate isintroduced in the amount of 1.5 to 2mg per kilogram of body weight overeach minute as in continuous infusion.
 9. A method of protectingpatients during operative procedures against unforeseen catastrophichypotension or hypoxia by introducing fructose-1,6-diphosphate in theamount of 50mg per kilogram of body weight.
 10. The method according toclaim 9, wherein fructose1,6-diphosphate is introduced in the amount of1.5 to 2mg per kilogram of body weight over each minute as in continuousinfusion.
 11. A method of conducting a barbiturate anesthesia with thehelp of fructose-1,6-diphosphate wherein said drug is introduced in thedose of 100-250mg per kilogram of body weight in a single dose.
 12. Themethod according to claim 11, wherein in an hourfructose-1,6-diphosphate is introduced in the dose of 2-3mg per kilogramof body weight per minute as in continuous infusion.